Members of the cytochrome P-450 (CYP) family of hemoproteins metabolise a wide variety of endogenous substrates such as steroid hormones, and of xenobiotics including carcinogens, toxins and drugs (1,2). Of the human CYP proteins, those of the CYP3A subfamily are of a major importance, since collectively, they are by far the most abundant of all the human CYP isoforms. Moreover, their substrate specificity is extremely broad; accordingly, many structurally diverse compounds are, exclusively or to some extent, substrates for CYP3A proteins. Based on the data available it is generally assumed that all CYP3A isoforms have similar substrate spectra; however, limited studies indicate the possibility of differences (3). All CYP3A isoforms are localized in organs of particular importance to drug disposition (gastrointestinal tract, kidney and liver).
At least three functional CYP3A proteins exist in humans. The CYP3A4 monooxygenase is the predominant cytochrome P450 in human liver and small bowel. The protein displays a broad substrate specificity and it metabolises more than 60% of all drugs that are currently in use, including contraceptive steroids, antidepressants, benzodiazepines, immunosuppressive agents, imidazole antimicotics, and macrolide antibiotics (4,5). In addition, CYP3A4 plays a major role in the protection from environmental toxins. For example, the protein metabolizes aflatoxin B1, which has been implicated in the etiology of liver cancer, which is a major cause of premature death in many areas of Africa and Asia. Aflatoxin B1 is a mycotoxin produced by species of Aspergillus, and human exposure results principally from the ingestion of stored foodstuffs contaminated with the mold. Carcinogenicity is associated with its conversion to 8,9-oxide by the hepatic cytochrome P450-dependent monooxygenase system. Forrester et al. (6) found that the rates of metabolic activation of aflatoxin B1 were highly correlated with the level of proteins of the CYP3A gene family in the microsomes. Furthermore, Paolini et al. (7) found significant increases in CYP3A in the lungs of rats treated with high doses of beta-carotene. Consequently, it was proposed that correspondingly high levels of CYP3A4 in humans would predispose an individual to cancer risk from the bioactivated tobacco-smoke procarcinogens, thus explaining the cocarcinogenic effect of beta-carotene in smokers. All this implies that individual variation in the CYP3A4 activity could influence the efficacy of a variety of drug therapies as well as the individual predisposition to several major cancers caused by environmental carcinogens.
A considerable variation in the CYP3A4 content and catalytic activity has been, indeed, described in the general population. For example, the metabolic clearance of the gene substrates exhibits a unimodal distribution with up to 20-fold interindividual variability. The activities of the CYP3A4 protein in liver biopsies vary up to 30-fold (8). Furthermore, many common drugs alter the expression levels of the gene (induction or repression) and the extent of this phenomenon is individually variable. The inducers of CYP3A4 expression include commonly used drugs such as the glucocorticoid dexamethasone, the antibiotic rifampicin, and the antimycotic clotrimazole. The inducibility of CYP3A4 expression, combined with the diverse renge of substrates, creates a potential for potentially harmful drug interactions involving this isozyme in patients undergoing therapies with multiple drugs.
CYP3A3 is a very closely related isoform to CYP3A4 (>more than 98% cDNA sequence similarity), but it is not known whether this reflects a separate gene product or an allelic variant. By contrast, CYP3A5 is a gene distinct from CYP3A4 and it is expressed polymorphically both in the adult and fetal liver and in the kidney and intestine. In adult Caucasians, the mRNA and the protein were detected in the liver of 10 to 30% of samples, while the protein was detected in the kidney and intestine of 70% of subjects (Ref. (9) and references therein). A point mutation described in the CYP3A5 gene which possibly results in the synthesis of an unstable protein, may account for the polymorphic expression of this enzyme (9). CYP3A7 is the third functional CYP3A isoform. CYP3A7 was originally isolated from a fetal liver but it was subsequently found in 54% of adult livers (10).
Tests to estimate the inducibility and the activity of CYP3A isozymes in an individual patient would be of obvious relevance for the optimization of therapies with drugs which are their substrates, and for the prevention of the associated side effects. Direct estimates of the activities of CYP3A isozymes in liver biopsies are possible but impracticable for both ethical and cost reasons. The indirect in vivo tests of CYP3A4 activity such as the erythromycin breath test or the 6-β-hydroxycortisol test pose ethical problems such as the invasive administration of undesirable probe substances and they are obviously unsuited for routine testing. In addition, the lack of correlation between these tests questions their informative value regarding the CYP3A4 activity (11).
A major portion (83%) of the interindividual CYP3A4 variability has been attributed to genetic factors (12). The establishment of a genetic test for the activity of CYP3A4 and of the other CYP3A isozymes should be possible, assuming the prior identification of those factors. Genetic variance affecting the activity and the expression of CYP3A isozymes could be localized in the genes itself, or in one or more of their regulators. A comparison of the three originally published sequences of the best characterized CYP3A gene, CYP3A4, suggested the existence of polymorphisms affecting the amino acid sequence of the CYP3A4 protein (13). Unfortunately, this observation has not been, to our knowledge, confirmed in the general population. More recently, a polymorphism (CYP3A4-W) has been described in the nifedipine-specific response element of the CYP3A4 promoter (14). Its presence associates with a more advanced prostate tumor stage (14). Felix et al. (15) examined this polymorphism in 99 de novo and 30 treatment-related leukemias. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A, such as epipodophyllotoxins. These data suggest that individuals with the CYP3A4-W polymorphism may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. At present it is unclear if the polymorphism influences the expressivity or inductibility of the CYP3A4 protein. A first published analysis suggests that it has no effect on the basal expression level of CYP3A4 (8). A point mutation was described in the CYP3A5 (9), whereas no mutations have been reported in CYP3A7.
Experiments with amino acid exchanges artificially introduced into the CYP3A4 gene indicate that the function of the family members may be quite sensitive to amino acid exchanges (16-21). Besides amino acid exchanges, silent polymorphisms and those localized in untranslated or intronic sequences also could influence the expression level of these genes. Alternatively, such polymorphisms could serve as markers for nearby, unidentified polymorphisms. This effect is known as linkage, i.e. defined polymorphisms serve as markers for phenotypes that they are not causative for.
A major breakthrough in the understanding of the CYP3A expression and inducibility took place in 1998 when three research groups independently showed that the expression of CYP3A4 is regulated by a member of the orphan nuclear receptor family termed PXR (pregnane X receptor), or PAR (22-24). Upon treatment with inducers of CYP3A4, PXR binds to the rifampicin/dexamethasone response element in the CYP3A4 promoter as a heterodimer with the 9-cis retinoic acid receptor (RXR). Northern blot analysis detected most abundant expression of hPXR in liver, colon, and small intestine, i.e. in the major organs expressing CYP3A4. The available evidence suggests that human PXR serves as a key transcriptional regulator of the CYP3A4 gene. A recent report describes the induction of CYP3A7 mediated by PXR suggesting that all members of the family may be regulated by this common transcriptional activator (25).
It is clear that naturally occurring mutations, if they exist can have effects on drug metabolization and efficacy of therapies with drugs, in particular in cancer treatment. It is unknown, however, how many of such variations exist, and with what frequency and at what positions in the human CYP3A4 and CYP3A7 genes.
Accordingly, means and methods for diagnosing and treating a variety of forms of individual drug intolerability and inefficacy of drug therapy which result from CYP3A4 and/or CYP3A7 gene polymorphisms that interfere e.g., with chemotherapeutic treatment of diseases, in particular cancer, were hitherto not available but are nevertheless highly desirable.
Thus, the technical problem of the present invention is to comply with the needs described above.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.